Large Granular Lymphocyte Leukemia Research Articles

Atlas of Diagnostic HematologyMSalama, JTeruya‐Feldstein and MKremyanskaya, Elsevier, 2021

It is encouraging that books dealing with the basic techniques of diagnostic haematology continue to be published. The scope of this 400-page book is broad, encompassing morphology, cytogenetics, molecular genetics, immunophenotyping and to some extent, radiology. There are 19 chapters, 10 of which deal with haematological neoplasms. In addition to red-cell themes, there are also chapters on haemophilia and blood transfusion. The 40 contributors are all drawn from the USA. Illustrations are generally of high quality. Most chapters are accurate and authoritative. The inclusion of clinical photographs and case studies in several chapters is a strength that could be emulated, linking as it does the laboratory features with the clinical approach to the patient. Do I have any reservations? The manner of stating the magnification of microscopic images is inconsistent between chapters and units used for the elements of the blood count are also inconsistent. Granulocyte is sometimes used as a synonym for neutrophil; it is not. Up-to-date gene names are not always used and sometimes are inconsistent within a single chapter, for example, ABL and ABL1, and MLL and KMT2A. There are some curious errors: a page of illustrations stated to be high-performance liquid chromatography (HPLC) actually shows capillary electrophoresis; an image of ‘beta thalassaemia minor’ shows two nucleated red blood cells and appears consistent with thalassaemia intermedia; T-cell large granular lymphocytic leukaemia is included in a chapter on myeloid neoplasms; a PEARL stain makes an appearance as does a haematoxylin and eosin-stained peripheral blood smear; and TdT is said to mean ‘tonoplast dicarboxylate transporter’ (it does, but was not intended here). Fortunately these errors are infrequent and the book will be appreciated.

Emerging Role of T-cell Receptor Constant β Chain-1 (TRBC1) Expression in the Flow Cytometric Diagnosis of T-cell Malignancies.

T-cell clonality testing is integral to the diagnostic work-up of T-cell malignancies; however, current methods lack specificity and sensitivity, which can make the diagnostic process difficult. The recent discovery of a monoclonal antibody (mAb) specific for human TRBC1 will greatly improve the outlook for T-cell malignancy diagnostics. The anti-TRBC1 mAb can be used in flow cytometry immunophenotyping assays to provide a low-cost, robust, and highly specific test that detects clonality of immunophenotypically distinct T-cell populations. Recent studies demonstrate the clinical utility of this approach in several contexts; use of this antibody in appropriately designed flow cytometry panels improves detection of circulating disease in patients with cutaneous T-cell lymphoma, eliminates the need for molecular clonality testing in the context of large granular lymphocyte leukemia, and provides more conclusive results in the context of many other T-cell disorders. It is worth noting that the increased ability to detect discrete clonal T-cell populations means that identification of T-cell clones of uncertain clinical significance (T-CUS) will become more common. This review discusses this new antibody and describes how it defines clonal T-cells. We present and discuss assay design and summarize findings to date about the use of flow cytometry TRBC1 analysis in the field of diagnostics, including lymph node and fluid sample investigations. We also make suggestions about how to apply the assay results in clinical work-ups, including how to interpret and report findings of T-CUS. Finally, we highlight areas that we think will benefit from further research.

Clonal hematopoiesis in adult pure red cell aplasia

Idiopathic pure red cell aplasia (PRCA) and secondary PRCA associated with thymoma and large granular lymphocyte leukemia are generally considered to be immune-mediated. The PRCA2004/2006 study showed that poor responses to immunosuppression and anemia relapse were associated with death. PRCA may represent the prodrome to MDS. Thus, clonal hematopoiesis may be responsible for treatment failure. We investigated gene mutations in myeloid neoplasm-associated genes in acquired PRCA. We identified 21 mutations affecting amino acid sequences in 11 of the 38 adult PRCA patients (28.9%) using stringent filtering of the error-prone sequences and SNPs. Four PRCA patients showed 7 driver mutations in TET2, DNMT3A and KDM6A, and 2 PRCA patients carried multiple mutations in TET2. Five PRCA patients had mutations with high VAFs exceeding 0.3. These results suggest that clonal hematopoiesis by stem/progenitor cells might be related to the pathophysiology of chronic PRCA in certain adult patients.

Large granular lymphocyte leukaemia study at the University Hospital of Donostia

IntroductionLarge granular lymphocyte (LGL) leukaemia is considered a mature T-cell or natural killer (NK) cell neoplasm, characterised by a clonal proliferation of LGL.AimsTo analyse the characteristics and to establish (if...

Acquired pure red cell aplasia and T cell large granular lymphocytic leukaemia in patients with autoimmune polyglandular syndrome type 1

BackgroundPure red cell aplasia (PRCA) and large granular lymphocytic leukaemia (LGLL) are very rare complications of autoimmune polyendocrine syndrome type 1 (APS1). Here, we report a case of APS1 with PRCA and LGLL. Previous cases were reviewed, and possible mechanisms are discussed.Case presentationA 31-year-old female presented with anaemia and was diagnosed with PRCA in our centre. She also had hypoparathyroidism for 24 years, premature ovarian failure for 10 years, osteoporosis for 5 years, recurrent pneumonia with bronchiectasis for 4 years and chronic diarrhoea for 1 year. Boosted whole-exome analysis showed AIRE heterozygous mutations, confirming the diagnosis as APS1. LGLL was diagnosed during follow-up. The PRCA responded well to glucocorticoid. treatmentConclusionAIRE is causally related to the development of LGLL and consequent PRCA, which may be due to some immunological mechanisms.

Lack of Viral Load Within Chronic Lymphoproliferative Disorder of Natural Killer Cells: What Is Outside the Leukemic Clone?

Large granular lymphocyte leukemias (LGLL) are sustained by proliferating cytotoxic T cells or NK cells, as happens in Chronic Lymphoproliferative Disorder of Natural Killer cells (CLPD-NK), whose etiology is only partly understood. Different hypotheses have been proposed on the original events triggering NK cell hyperactivation and transformation, including a role of viral agents. In this perspective, we revise the lines of evidence that suggested a pathogenetic role in LGLL of the exposure to retroviruses and that identified Epstein Barr Virus (EBV) in other NK cell leukemias and lymphomas and focus on the contrasting data about the importance of viral agents in CLPD-NK. EBV was detected in aggressive NK leukemias but not in the indolent CLPD-NK, where seroreactivity against HTLV-1 retrovirus envelope BA21 protein antigens has been reported in patients, although lacking clear evidence of HTLV infection. We next present original results of whole exome sequencing data analysis that failed to identify viral sequences in CLPD-NK. We recently demonstrated that proliferating NK cells of patients harbor several somatic lesions likely contributing to sustain NK cell proliferation. Thus, we explore whether “neoantigens” similar to the BA21 antigen could be generated by aberrancies present in the leukemic clone. In light of the literature and new data, we evaluated the intriguing hypothesis that NK cell activation can be caused by retroviral agents located outside the hematopoietic compartment and on the possible mechanisms involved with the prospects of immunotherapy-based approaches to limit the growth of NK cells in CLPD-NK disease.

Single-Antibody Evaluation of T-Cell Receptor β Constant Chain Monotypia by Flow Cytometry Facilitates the Diagnosis of T-Cell Large Granular Lymphocytic Leukemia.

The diagnosis of T-cell large granular lymphocytic leukemia (T-LGLL) is challenging because of overlapping immunophenotypic features with reactive T cells and limitations of T-cell clonality assays. We studied whether adding an antibody against T-cell receptor β constant region 1 (TRBC1) to a comprehensive flow cytometry panel could facilitate the diagnosis of T-LGLL. We added TRBC1 antibody to the standard T-cell and natural killer (NK) cell panel to assess T-cell clonality in 56 T-LGLLs and 34 reactive lymphocytoses. In addition, 20 chronic lymphoproliferative disorder of NK cells (CLPD-NKs) and 10 reactive NK-cell lymphocytoses were analyzed. Clonal T cells were detected in all available T-LGLLs by monotypic TRBC1 expression and clonal/equivocal T-cell receptor gene rearrangement (TCGR) studies, compared with only 27% of T-LGLLs by killer-cell immunoglobulin-like receptor (KIR) restriction. Overall, 85% of T-LGLLs had a blood tumor burden greater than 500 cells/µL. Thirty-four reactive cases showed polytypic TRBC1 expression, except for 5 that revealed small T-cell clones of uncertain significance. All CLPD-NKs showed expected clonal KIR expression and negative TRBC1 expression. Addition of TRBC1 antibody to the routine flow cytometry assay could replace the TCGR molecular study and KIR flow cytometric analysis to assess clonality, simplifying the diagnosis of T-LGLL.

Discovery of Dysimmunity in Large Granular Lymphocytic Leukemia (LGLL) using Bioinformatic Analysis.

Large granular lymphocytic leukemia (LGLL) is a chronic lymphoproliferative disorder characterized by the clonal proliferation of large granular lymphocytes (LGL), classified as T and NK subtypes. Although JAK/STAT pathway gene mutation, such as STAT3/STAT5B, is the dominant driver in the proliferation of LGLL, immune abnormality remains an unsolved puzzle in the pathogenesis. By means of bioinformatic method through the GEO dataset GSE39838, we performed the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, as well as protein-protein interaction network (PPI) module calculation. As a consequence, differentially expressed genes (DEGs) involved in immune regulation were detected to be related with LGLL, including C1QA, C1QC and CD163 etc. Among all the DEGs, 147 genes were up-regulated, while the number of down-regulated genes was 1,296. In the KEGG pathway of LGLL, infection and immunity were the primary alteration, including tuberculosis and rheumatoid arthritis (RA). However, meticulous experiments are required to validate. To sum up, dysimmunity might be another internal anomaly of LGLL, thus it is a reminder that immune regulation of LGLL should be paid more attention. Moreover, immune microenvironment studies in LGLL covering T, B, and NK cells probably contribute to the molecular pathology, aiming to contribute to the molecular pathology of the LGLL. Additionally, pharmaceutical development directed at immune molecules might be pre-dictive of targeted therapy era in LGLL.

Chronic urticaria versus dermatomyositis in a case of T- cell large granular lymphocytic leukemia

Aim: The reported case involved a complicated diagnostic path, not only because of the usual difficulties specific to chronic urticaria but, also because the trigger was a T-cell large granular lymphocytic leukemia (T-LGLL), a rare type of leukemia with an indolent course whose etiology is still not well known. This leukemia is also known for its propensity to cause autoimmune diseases. The aim of this study was to identify whether the muscle damage was caused by dermatomyositis or by T-LGLL. Methods: After elevated muscle enzyme levels had been discovered magnetic resonance imaging (MRI) revealed muscle damage. Consequently, a muscle biopsy was performed in a targeted manner. In addition to muscle biopsy, transmission electron microscopy and anti-CN1A antibody testing were performed. Results: MRI of the lower limbs and pelvic girdle indicated moderate fibroadipose substitution in many muscles, moderate edema in others. No involvement of the shoulder girdle and upper limbs. Histological examination of the muscle fibers showed an “inflammatory myopathy with isolated phagocytotic fibers.” Inclusion-body myositis, which is known to be associated with chronic T-LGLL was excluded. On the same biopsy transmission electron microscopy confirmed inflammatory myopathy and anti-CN1A antibodies were positive. DNA extracted from the muscle of the micro-rearrangement for the y-chain of the TCR identified on the DNA extracted from peripheral blood was positive. Conclusions: Conclusions: Chronic urticaria was an indication of immunoproliferative disease. Myositis was the pathology due to T-LGLL, and dermatomyositis was excluded.

Diagnosis and management of pure red cell aplasia

Pure red cell aplasia (PRCA) is characterized by normocytic anemia with reticulocytopenia and marked reduction of the bone marrow erythroid precursors. PRCA may be congenital (Diamond-Blackfan anemia) or acquired. Acquired PRCA may present in the context of various backgrounds, the most common type in Japan being idiopathic, thymoma-associated, and large granular lymphocyte leukemia. Idiopathic and secondary PRCA that do not respond to the treatment of the underlying disease are generally treated using immunosuppressive agents. A retrospective study PRCA2004/2006 suggests that maintenance therapy and management of infectious complications is crucial for improving the prognosis in patients with PRCA. Recently, allogeneic stem cell transplantation has been considered as a potential option for the treatment of patients with PRCA who are refractory to immunosuppressive therapy. Sirolimus and roxadustat may be effective for relapsed/refractory PRCA with renal insufficiency and anti-erythropoietin antibody-mediated PRCA, respectively. Some gene mutations were detected in certain patients who had acquired PRCA, and the identification of STAT3 mutations may be useful in PRCA management. A prospective cohort study PRCA2016 has been ongoing in Japan, and novel discoveries provide hope for improving the outcome in patients with PRCA.

Thymoma-associated multiorgan autoimmunity with cutaneous only presentation: A case report.

Thymoma-associated multiorgan autoimmunity disease can be seen in patients with thymomas and presents with features of graft versus host disease. Here, we report a case of a 52-year-old woman with a complex medical history including malignant thymoma, myasthenia gravis, Good’s syndrome, and T cell large granular lymphocytic leukemia who presented with a diffuse pruritic rash ultimately found to be compatible with a cutaneous presentation of thymoma-associated multiorgan autoimmunity disease. The eruption heralded the recurrence of the malignant thymoma and the rash was ultimately found to be resistant to a multitude of therapies except for oral corticosteroids.

Large Granular Lymphocytosis With Cytopenias After Allogeneic Blood or Marrow Transplantation: Clinical Characteristics and Response to Immunosuppressive Therapy

Large granular lymphocytosis (LGL)-or LGL leukemia-is a T- or NK-cell lymphoproliferative disorder that often results in cytopenias and autoimmune phenomena. Several studies have described LGL in a subset of patients after allogeneic blood or marrow transplantation (alloBMT), almost exclusively in the setting of asymptomatic lymphocytosis. Some have suggested an association with improved transplant-related outcomes. In contrast, clinically significant LGL after alloBMT is only described in small case reports. This study sought to assess the characteristics, significance, and response to treatment of LGL associated with unexplained anemia, thrombocytopenia, or neutropenia after alloBMT. We performed a retrospective analysis of 150 patients who were evaluated for LGL by peripheral blood flow cytometry (LGL flow) for unexplained cytopenias following initial engraftment after alloBMT from January 1 2012 to July 1, 2019. We identified patients with abnormally increased populations of LGL cells (LGL+) as assessed by Johns Hopkins Hematopathology. We collected demographic, transplantation, and LGL treatment information from electronic medical records. We compared LGL+ patients to patients with unexplained cytopenias with negative flow cytometry for LGL (LGL-) in this cohort. We also assessed change in blood counts after 4 weeks of immunosuppressive therapy in LGL+ patients. Cytopenias occurred at a median of 5.7 months (range 1-81) after alloBMT. The majority of the transplants were nonmyeloablative from haploidentical donors, and all patients received post-transplantation cyclophosphamide for graft-versus-host disease prophylaxis, consistent with the overall alloBMT characteristics at our center. We identified 70 patients with LGL and cytopenias, representing 47% of those evaluated by flow cytometry. There were no significant demographic or transplant-related differences between LGL+ patients and LGL- patients. The median age was 59, and 63% were male. LGL+ patients were more likely to have had cytomegalovirus (CMV) viremia (73% versus 28%, P < .0001), but not acute or chronic graft-versus-host disease. LGL+ patients had higher absolute lymphocyte counts (1500 versus 485/ mm3, P < .0001), a trend toward lower absolute neutrophil count (660 versus 965/mm3, P=.17), and lower neutrophil to lymphocyte ratio (0.39 versus 1.71, P < .001). There were no differences in overall survival or relapse-free survival. Of those with T-cell LGL, 45 were assessed for T-cell receptor clonality. In all, 22% were clonal, 53% oligoclonal, 4% polyclonal, and 20% indeterminate. Thirty (43%) LGL+ patients received immunosuppressive therapy (IST) for cytopenias. First-line treatment was corticosteroids for 25 (83%). Among those treated, there was an increase in median absolute neutrophil count from 720 before treatment to 1990/mm3 after 4 weeks (P=.0017). Thrombocytopenia and anemia showed at most a mild improvement with IST. LGL was a common association with otherwise unexplained cytopenias after alloBMT, almost always after prior CMV infection. LGL in the setting of cytopenias did not predict improved transplantation outcomes compared to those with cytopenias without presence of LGL. IST was effective at improving neutropenia associated with LGL after alloBMT.

T Cell Receptor (α, β, γ) Gene Rearrangements and Expression in Normal and Leukemic Large Granular Lymphocytes/Natural Killer Cells

The large granular lymphocyte (LGL) population, which effects a natural killer (NK) function, consists of cells whose lineage derivation has not been clearly established on the basis of phenotypic and functional properties. To clarify the relationship of LGL/NK cells to T cells we studied patterns of rearrangement and expression of the T cell receptor (Ti) genes alpha, beta, and gamma in normal human LGLs; in CD8+, CD8-, Mol+, and Mol- LGL subsets; and in 17 cases of leukemic LGL proliferations (T gamma LPD). T alpha, T beta, and T gamma genes were not expressed, nor were T beta and T gamma genes rearranged in normal LGLs or LGL subsets. The T gamma LPD were divided into two groups. One group (15/17 cases) was characterized as CD3+ and displayed Ti gene rearrangements. Seven of these cases were reactive with monoclonal antibody WT31, which suggested expression of an alpha/beta heterodimer on the cell surface. The other group (2/17 cases) was CD3- with unrearranged Ti genes. These results indicate that the normal LGL/NK population is homogeneous and distinct from the normal T cell population because it does not express, and as a result, cannot effect its immune function through the T cell receptor molecules. Conversely, T gamma LPDs represent a heterogeneous group of lymphoproliferative diseases within which the CD3-, Ti- cases most likely represent the neoplastic counterpart of normal LGL cells. The more frequent CD3+ cases may be related to recently described NK-like T cells. The observations that normal LGLs maintain germline T gamma genes and that many CD3+ T gamma LPD display an alpha/beta heterodimer suggest that a T gamma-containing receptor may not be necessary for NK or NK-like cytotoxicity.

Analysis of a single-institution cohort of patients with Felty's syndrome and T-cell large granular lymphocytic leukemia in the setting of rheumatoid arthritis

T-cell large granular lymphocytic leukemia (T-LGLL) is a lymphoproliferative disorder characterized by a persistent increase in the number of large granular lymphocytes (LGLs), neutropenia, and splenomegaly. Clinical manifestations of T-LGLL in the setting of rheumatoid arthritis (RA) are often identical to those in which one would suspect Felty's syndrome (FS). These disorders are distinguished by the presence of T-cell clonality, which is present in T-LGLL but not in FS. Mutations in the signal transducer and activator of transcription 3 (STAT3) and 5b (STAT5b) genes can be used as molecular markers of T-LGLL, but their prevalence in FS is unknown.Eighty-one patients with RA and unexplained neutropenia or/and an increase in the number of LGLs above 2 × 109/L were stratified into RA-associated T-LGLL (N = 56) or FS (N = 25) groups based on the presence or absence of T-cell clonality. STAT3 and STAT5b gene mutations were assessed in each group by means of allele-specific polymerase chain reaction assays. Clinical, immunological, laboratory data and the results of immunophenotyping of blood and bone marrow lymphocytes were also evaluated.Mutations of the STAT3 gene and an increase in the number of LGLs above 2 × 109/L were detected in RA-associated T-LGLL, but not in FS (39% vs 0% and 21% vs 0%, respectively). Mutations in the STAT5b gene were not observed in either group. Expression of CD57, CD16, and CD5−/dim on CD3+CD8+ T-lymphocytes was observed in both RA-associated T-LGLL and FS.STAT3 gene mutations or LGL counts over 2 × 109/L in RA patients are indicative of T-LGLL.

STAT3 and STAT5B Mutations in T/NK-Cell Chronic Lymphoproliferative Disorders of Large Granular Lymphocytes (LGL): Association with Disease Features.

Simple SummarySTAT3 and STAT5B mutations have been identified in a subset of T and NK large granular lymphocytic leukemia (T/NK-LGLL). The aim of our study was to evaluate the frequency and type of these mutations in all different subtypes of T/NK-LGL expansions (n = 100 patients), as well as to analyze its association with biological and clinical features of the disease. We show for the first time that STAT3/5B mutations were present in all different T/NK-cell LGLL categories here studied; further, STAT3 mutations were associated with overall reduced counts of almost all normal residual populations of immune cells in blood, together with a shorter time-to-therapy vs. wild type T/NK-LGLL. These findings contribute to support the utility of the STAT3 mutation analysis for diagnostic and prognostic purposes in LGLL.STAT3 and STAT5B (STAT3/STAT5B) mutations are the most common mutations in T-cell large granular lymphocytic leukemia (T-LGLL) and chronic lymphoproliferative disorders of NK cells (CLPD-NK), but their clinical impact remains unknown. We investigated the frequency and type of STAT3/STAT5B mutations in FACS-sorted populations of expanded T/NK-LGL from 100 (82 clonal; 6 oligoclonal; 12 polyclonal) patients, and its relationship with disease features. Seventeen non-LGL T-CLPD patients and 628 age-matched healthy donors were analyzed as controls. STAT3 (n = 30) and STAT5B (n = 1) mutations were detected in 28/82 clonal T/NK-LGLL patients (34%), while absent (0/18, 0%) among oligoclonal/polyclonal LGL-lymphocytosis. Mutations were found across all diagnostic subgroups: TCD8+-LGLL, 36%; CLPD-NK, 38%; TCD4+-LGLL, 7%; Tαβ+DP-LGLL, 100%; Tαβ+DN-LGLL, 50%; Tγδ+-LGLL, 44%. STAT3-mutated T-LGLL/CLPD-NK showed overall reduced (p < 0.05) blood counts of most normal leukocyte subsets, with a higher rate (vs. nonmutated LGLL) of neutropenia (p = 0.04), severe neutropenia (p = 0.02), and cases requiring treatment (p = 0.0001), together with a shorter time-to-therapy (p = 0.0001), particularly in non-Y640F STAT3-mutated patients. These findings confirm and extend on previous observations about the high prevalence of STAT3 mutations across different subtypes of LGLL, and its association with a more marked decrease of all major blood-cell subsets and a shortened time-to-therapy.

Large Granular Lymphocytic Leukemia with Coexisting Malignancies

Introduction: Large granular lymphocytic leukemia (LGLL) is a rare, indolent malignancy arising from cytotoxic T cells (~85%) or NK cells (~15%). The coexistence of LGLL with MDS, B cell neoplasms and plasma cell dyscrasias have been reported in case reports and two retrospective studies (Gayal et al 2018, Viny et al 2008). However, those studies were limited by sample size and a small number of coexisting malignancies. To our knowledge, there is no systematic study on coexisting solid tumors with LGLL in the literature. In present study, we systemically analyzed the coexisting malignancies (CMs) including solid tumors and hematological malignancies (liquid tumors) in our LGLL patients (pts). We compared the clinical features, immunophenotype, treatment responses and estimated outcomes of CM-associated LGLL and non-CM associated LGLL pts. Methods: Total 303 consecutive LGLL pts evaluated at Moffitt Cancer Center (MCC) between 2001 and 2017 were included in the study. Pts who did not meet the WHO diagnostic criteria of peripheral blood (PB) LGL count ≧ 2.0 k/μL but presented with persistent LGL clonal populations along with characteristic immunophenotype, matching clinical features and bone marrow involvement with clonal LGL population were considered to have LGLL. Results: Among the total 303 LGLL patients, 123 (40.6%) had at least 1 CM. The maximum number of coexisting malignancies were 3. 114 pts (37.6%) had invasive cancers (excluding non-melanomatous skin cancer), 83 (27.1%) had solid tumors and 60 (19.8%) had liquid tumors. The most frequent invasive solid tumors in our LGLL patients were: prostate cancer, breast cancer, lung cancer, colorectal cancer, and melanoma. The most frequent liquid tumors were: B-cell lymphoma, T-cell lymphoma, CLL/SLL, CML and MM (Table 1). The number of patients with CM diagnosed before, at the same time and after LGLL diagnosis were 103, 7, and 28, respectively. Table 2 compared the clinical features of LGLL pts with CM to those without CM separated into liquid and solid tumors. Compared to pts without CM, LGLL patients with liquid tumors were older, less likely to have B symptoms (49.2% vs 73.6%, respectively), less likely to be neutropenic (32.1% vs 53.4%), less likely to be pRBC transfusion dependent (7.3% vs 24.5%), and less likely to require treatment (33.3% vs 61.1%). LGLL pts with solid tumors compared to those without CM were older, more likely to be Caucasians, more likely to have NK-cell LGLL (13.3% vs 6.1%), and less likely to have B symptoms (59.5% vs 73.6%). NGS testing was performed in 25 patients. Activating STAT3 mutations were detected in 9/21 patients without CM and 1/4 patients with CM (p=0.63 by Fisher's exact test). None of tested pts demonstrated STAT5B mutation. An extensive 10-color flow cytometry panel was employed after year 2015 in 34 pts. The immunophenotype was similar between CM-associated T-LGLLs and non-CM associated T-LGLLs except that CM-associated T-LGLL pts were more likely to be CD56+ (6/11 vs 3/23, p=0.03 by Fish's exact test). CM-associated LGLLs and non-CM associated LGLLs had comparable response rates to treatments (Table 3). The median follow-up time for all patient population was 4.8 years. Pts with CM had worse overall survival (OS) (Figure 1). The survival difference was significant after adjusting for age, gender and race (data not shown). Conclusions: In the present study of 303 consecutive LGLL pts, 40.6% had CM, including 27.4% with solid tumors and 19.8% with liquid tumors. CM-associated LGLLs had similar clinical and pathologic features and treatment responses as non-CM associated LGLLs, except that LGLL pts with CM were less likely to be symptomatic and less likely required treatment. Earlier detection of LGLL in pts with liquid tumors may have contributed to those differences. Interestingly, T-cell LGLLs with CM were more likely to be CD56 positive and LGLLs associated with solid tumors were more likely to be derived from NK-cell lineage. The high incidence of CMs in LGLL can be explained by the following hypotheses: 1) CM provided an initial antigenic stimulus and contributed to the pathogenesis of LGLL or alternatively, 2) host immunosuppression due to clonal LGLL proliferation promoted development of CM. The findings in this study that a majority of CMs manifested prior to LGLL diagnosis favor the first hypothesis. Our results provide additional insights into the pathogenesis of LGLL. Disclosures Komrokji: BMS: Honoraria, Speakers Bureau; Agios: Speakers Bureau; Novartis: Honoraria; Acceleron: Honoraria; Incyte: Honoraria; Abbvie: Honoraria; Geron: Honoraria; Jazz: Honoraria, Speakers Bureau. Sokol:EUSA Pharma: Consultancy, Honoraria, Speakers Bureau; Kyowa/Kirin Inc.: Membership on an entity's Board of Directors or advisory committees; Kymera Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Analysis of Genomic Landscape of Large Granular Lymphocyte Leukemia Reveals Etiologic Insights

Introduction: Large Granular Lymphocyte (LGL) leukemia is a rare lymphoproliferative disorder characterized by clonal expansion of either CD3+ cytotoxic T cells expressing T cell receptor (TCR) alpha beta or CD3- natural killer (NK) cells. A less frequent CD3+ T cell subtype expresses TCR gamma delta (GD). Here, we present the molecular landscape of known LGL types (T, NK, GD) from analysis of the largest patient cohort assembled to date. An integrative analysis of genomic datasets from all LGL subtypes is necessary to more precisely define the shared and unique etiology of this rare disorder. Methods: We collected paired saliva and PBMC samples and related clinical information from 116 LGL leukemia patients after informed consent. The assembled cohort consisted of 93 T-LGL, 11 NK-LGL, and 12 GD T-LGL patients. Genomic analyses were performed on the leukemic (PBMC) and germline (saliva) samples after whole exome sequencing (WES) and transcriptome sequencing (RNAseq, PBMC only). Results: Somatic mutations were detected in the previously described potential drivers STAT3 (n=56), TNFAIP3 (n=9), and PIK3R1 (n=4). We also identified somatic mutations in CDH8 (n=3) and CCL22 (n=4), which we postulate as putative drivers based on mutational clustering. CDH8 was mutated in all three LGL subtypes, but CCL22 somatic mutations were only observed in NK-LGL patients. We observed that STAT3 and CCL22 together account for 64% (7/11) of NK-LGL cases. STAT3 is the most recurrently mutated gene in LGL leukemia, yet concurrent molecular and clinical features are incompletely defined. Interestingly, patients with STAT3 mutations have a higher mutational burden (P=0.0006) compared to those with wild-type (WT) STAT3. This effect is independent of the age of the patient, which correlates with the mutational burden (R=0.26, P=0.0039) and agrees with the finding that the dominant mutational signatures in this cohort exhibit clock-like properties. We also observed that patients with STAT3 mutations are enriched (P=0.0273) for additional mutations in chromatin modifier enzymes such as KMT2D, TET2, DNMT3A, and SETD1B (Figure 1). We found that ~10% of the samples exhibit broad somatic copy-number aberrations, and a patient with somatic mutations in STAT3 and KMT2D displayed high-level microsatellite instability. STAT3 mutations were also significantly associated with increased expression of genes involved in apoptosis, complement activation, and interferon cytokine signaling compared to STAT3 WT (FDR &amp;lt; 0.05). Early-onset LGL patients with age 51 years or less (n=28), as defined by the bottom quartile of the cohort, displayed no differential enrichment of the somatic driver genes. Interestingly, the age of the patient was significantly associated with absolute neutrophil counts (ANC) (P = 0.0068), with younger patients exhibiting lower neutrophil counts, even after adjusting for the presence of STAT3 mutation, as it is associated with lower ANC. As neutropenia is a hallmark feature of LGL leukemia and often a trigger for initiating therapy, the association of young age with lower neutrophil counts and lower somatic mutational burden suggests other mechanisms may be involved. Focusing on the germline variants, we found that 17 of the patients (14.6%) had at least one pathogenic or likely pathogenic germline variant with known oncogenic association as annotated using CharGer. 5 patients had pathogenic mutations in known tumor suppressors including FANCC (n=1), BRCA1 (n=1), PALB2 (n=1), MUTYH (n=1), and SDHA (n=1), while 1 patient had pathogenic mutations in ALK, a known oncogene. Conclusions: We report on the genomic analyses done on whole exome and RNA-seq data from the largest cohort assembled for LGL leukemia to date. We show that the presence of STAT3 mutation is significantly associated with an increase in mutation burden and additional somatic mutations in chromatin modifiers, hinting at potential pathogenic mechanisms within STAT3 mutated patients. By combining LGL subtypes in our analysis, we were able to identify CDH8 as a putative driver that is present in T, NK, and GD subtypes. Additionally, we report CCL22 mutations specific to NK-LGL leukemia, however did not detect any subtype specific mutations in GD T-LGL. We found that about 15% of the patients carry at least one pathogenic germline variant with known oncogenic associations. These findings highlight emerging etiologic insights into this rare disorder. Disclosures Feith: Kymera Therapeutics: Membership on an entity's Board of Directors or advisory committees. Loughran:Keystone Nano: Membership on an entity's Board of Directors or advisory committees; Bioniz Therapeutics: Membership on an entity's Board of Directors or advisory committees; Kymera Therapeutics: Membership on an entity's Board of Directors or advisory committees; Dren Bio: Membership on an entity's Board of Directors or advisory committees.

Thalidomide Plus Prednisone and Methotrexate for Symptomatic Large Granular Lymphocyte Leukemia: A Prospective, Single-Center, Pilot Study

Background: Large granular lymphocyte leukemia (LGLL) is one type of chronic lymphocytic proliferative disorders, which commonly manifests as infiltration of large granular lymphocytes in both peripheral blood and bone marrow. LGLL now includes two entities with similar clinical course, treatment strategy and outcomes: T-cell large granular lymphocytic leukemia and chronic lymphoproliferative disorder of NK cells. The standard therapy for LGLL is still elusive. Here, we presented the efficacy and safety of combinatorial oral immunoregulatory regimen thalidomide, prednisone, and methotrexate (TPM regimen) in a prospective phase 2 clinical trial. Methods: We designed this phase 2 investigator-initiated clinical trial (NCT04453345) to evaluate the clinical response and safety of the combination of thalidomide, prednisone, and methotrexate in symptomatic treatment naïve LGLL patients. The TPM regimen includes thalidomide 50-100mg per night, prednisone 0.5-1.0mg/kg qod and methotrexate 10mg/m2 per week. This regimen will be administrated for up to 12 months until disease progression or intolerable. Then, thalidomide maintenance will continue for another year or until intolerance. Meanwhile, we set Cyclosporin A (CsA) alone or plus steroids as control. Treatment dosage for CsA was 3-5mg/Kg/day with or without steroids (prednisone) 0.5-1 mg/Kg/day. The primary endpoint of this study was the complete response rate. Results: From Aug 2013, to Jan 2020, twenty-eight patients were enrolled in this study. The median follow-up time was 26 months (range: 7-96). Twenty-five patients (89%) achieved hematologic and symptomatic response. Among them, 21 patients (75%) achieved complete response (CR) and four patients achieved partial response. The median time to best clinical response was 6 months (2-18). The 3-years progression-free-survival (PFS) rate was 90%, and 3-years overall survival (OS) rate was 92%. The median PFS time was not reached in TPM group. The curative effect was better for TPM treatment group, both for overall response (OR) (TPM 89% (25/28) vs CsA 49% (49/99), P=0.000) and CR (TPM 75% (21/28) vs CsA 20% (20/99), P=0.000). Adverse events were uncommon, two patients had grade 1-2 nausea and one had grade 3 nausea. Two patients had grade 1-2 constipation and one patient experienced grade 1-2 peripheral neuritis. Conclusion: The efficacy of this TPM regimen is higher than the history reports with limited adverse events. The multiple-center clinical trial has been initiated to validate this conclusion. Disclosures No relevant conflicts of interest to declare.

Myelodysplastic Syndromes (MDS) & COVID-19: Clinical Experience from the US Epicenter of the Pandemic

Background:The COVID-19 (SARS-CoV-2) pandemic has affected cancer patients (pts) in a myriad of ways, including diagnostic & treatment delays, scarcity of blood products, and most importantly, higher risks of morbidity and mortality from the viral infection itself. Though COVID-19’s effects on many specific cancers has previously been described, there has been little reported on its effects on pts with MDS.Methods:We prospectively reviewed the records of all pts seen in the MDS clinic of a large New York City tertiary academic medical center between March 12 and May 07 2020. A confirmed case of COVID-19 was defined by a positive (+) result on a real-time reverse-transcriptase polymerase chain reaction (PCR) assay of a specimen collected on a nasopharyngeal swab, or detectable COVID-19 antibodies. COVID-19 antibodies were determined via an IgG assay developed at Mount Sinai with the ability to detect antibody titers to a dilution of 1:2880. Initially, only symptomatic pts were screened due to limited testing availability. However, after April 7, all clinic pts were screened by PCR.Results:Among 85 pts seen in the clinic, 23 were found to have COVID-19 (27.1%). The median age of all pts was 72 years (range 20-90); racial breakdown included 58.8% Caucasian, 12.9% Hispanic, 10.6% African-American, 7.1% Asian, and 10.6% other. Of note, 65.2% of COVID-19+ pts were Caucasian, 13.0% Hispanic, and 17.4% other. The most common diagnoses were MDS (n=61), AML (n=10), multiple myeloma (MM) (n=9), large granular lymphocytic leukemia (LGL) (n=6), ALL (n=6), MPN (n=4), NHL (n=3), and CML (n=2). Forty-two of the MDS patients had no other malignancies; co-diagnoses among the remaining MDS patients included MM (n=8), LGL (n=6), & T-cell dyscrasias (n=5).Of the 23 COVID-19+ pts, 11 (47.8%) were hospitalized and 4 (17.4%) were asymptomatic. Common symptoms were URI symptoms (15) and fever (14). ARDS and pneumonia occurred in 4 pts. The most common treatments were hydroxychloroquine (n=4), steroids (n=3), and azithromycin (n=2). Eighteen of the 23 + pts (78.3%) had MDS; the others included pts with MPN, APL, ALL, AML, or NHL. Among the COVID-19+ MDS pts, IPSS-R was very low (n=5), low (n=3), intermediate (n=1), high (n=2), and very high risk (n=5) in evaluable patients. MDS directed treatment included azacitidine (8) (2 with venetoclax), erythropoietin stimulating agents (3), best-supportive care (4), intravenous immunoglobulin (3), and lenalidomide (1). Disease status at diagnosis was stable disease (SD; n=9), hematologic improvement (HI; n=6), progressive disease (PD; n=1), and complete response (CR; n=1). The median number of co-morbidities per pt was 2. Major co-morbidities included cardiac disease (n=5), hypertension (n=5), diabetes (n=5), and dementia (n=2). COVID-19+ pts remained PCR+ for a range of 14-42 days in those with available serial testing (n=8). Of the PCR+ pts, 14 were tested for the presence of COVID-19 antibodies; 12 were +, with a range of titers from <1:80 to 1:2880. Three patients were PCR negative at least once but tested antibody positive.Nine of 23 (39%) COVID-19+ pts died; mortality among MDS patients included 6/18 (33%) COVID-19+ and 6/61 (9.8%) from the overall cohort. Among the 9 who died, the median number of co-morbidities was 3 and the median age was 75 years-old. Disease status in these patients was SD (2), CR (1), HI (1), and PD (1). IPSS-R was very high (n=4), high (n=1), and intermediate (n=1) in the MDS pts who died.Conclusions:This represents the first reported large case series regarding the risks of developing COVID-19 and its effects at an MDS clinic in the initial US epicenter of the pandemic. Overall, 27.1% of the pt population was diagnosed with COVID-19; 39.1% of these pts died, or 10.6% of the overall cohort. A retrospective study across the US reported a 16% mortality rate of COVID-19 in cancer pts (Dr Rivera et al, 2020). The mortality rate reported here is higher, but with a smaller sample size. The clinical significance of persistently + PCR tests up to 6 weeks is unclear. COVID-19 antibodies were found in 85.7% of COVID-19 PCR+ pts tested, showing MDS pts can mount a humoral response. Likely factors contributing to the high mortality of MDS pts were co-morbidities and age. The majority of pts recovered and have resumed MDS directed therapy. Protecting MDS pts from COVID-19 infection must be a primary overall therapeutic approach until there are more effective COVID-19 treatment strategies.DisclosuresNavada:Onconova Therapeutics Inc: Research Funding. Silverman:Medimmune: Research Funding; Onconova Therapeutics Inc: Patents & Royalties, Research Funding; Celgene: Research Funding.

Germline Gain-of-Function JAK3 Mutation in Familial Chronic Lymphoproliferative Disorder of NK Cells

Chronic lymphoproliferative disorder of NK-cells (CLPD-NK) predominantly occurs in adults with a median age of diagnosis at 60 years. It is characterized by a persistent increase (≥2 x 109/L, for &amp;gt; 6 months) of mature NK-cells in peripheral blood with an indolent clinical course similar to T-cell large granular lymphocytic leukemia (T-LGL). Somatic gain-of-function (GOF) mutations in STAT3 have been identified in approximately one-third of patients with CLPD-NK. On the other hand, somatic GOF mutations in JAK3 recurrently occur in various types of T-cell neoplasms and exert a GOF effect, unlike biallelic germline loss-of-function mutations found in severe combined immunodeficiency (Figure 1). Here we report on the discovery of a germline GOF JAK3 mutation as a first germline cause of CLPD-NK. Two individuals from one nonconsanguineous family (mother and son) presented at ages 35 and 12 years old with NK cell lymphoproliferation, lymphadenopathy, splenomegaly and autoimmune symptoms. The mother had history of vasculitis while the son was diagnosed with CVID, recurrent multilineage autoimmune cytopenia and subsequently developed psoriasis at 18 years old. The immunological phenotype was assessed in depth in the son and revealed hypogammaglobulinemia with normal vaccine response, expanded NK cells (between 40-60% of total lymphocytes), decreased FOXP3 expression in regulatory T cells and B cell subsets showing decreased total and isotype-switched memory B cells. Flow cytometry revealed expanded population of aberrant NK cells with normal KIR panel. Marrow studies revealed normal karyotype, cellularity and maturation but prominent large granular lymphocytes with benign cytology. Genomic studies identified a novel germline heterozygous JAK3 variant (c.1520A&amp;gt;C/p.Q507P) located at the linker between SH2 and pseudokinase domain (Figure 1). No additional somatic mutations were found. The JAK3 variant was not present in gnomAD database but previously reported as somatic mutation in a patient with T cell prolymphocytic leukemia (Bergmann, Genes Chromosomes Cancer 2014) and predicted to exert a GOF effect. It is well known that JAK3 activation promotes STAT signaling, a known key player in lymphoproliferation. To better understand the biological effect in patient cells, we performed pSTAT5 phosphorylation assay in primary blood lymphocytes after IL2 stimulation, revealing increased pSTAT5 phosphorylation in patient's NK cells. The IL3-dependent BaF3 cell line (containing human wild type JAK3) has been previously used as a robust model to study the effect of JAK3 mutations (Elliott et al. Blood 2011). We therefore introduced the p.Q507P mutation using CRISPR/Cas9 system and used known GOF mutation p.A572V as positive control. While untransduced BaF3 cells died without IL3, p.Q507P-mutant BaF3 cells survived and rapidly expanded without IL3, showing comparable results to positive control. Finally, using western blot we identified constitutive phosphorylation as expected mechanism underlying the lymphoproliferation p.Q507P-mutant cells. In summary, we identify JAK3 as the first germline cause underlying familial CLPD-NK and describe a novel primary immune dysregulatory disorder characterized by non-malignant NK lymphoproliferation with CVID and autoimmune dysregulation. These findings broaden the genetic spectrum of primary immunodeficiency and immune dysregulatory conditions. Disclosures Takemoto: Genentech: Membership on an entity's Board of Directors or advisory committees; Novartis: Other: DSMB Aplastic Anemia Trial. Nichols:Incyte corporation: Research Funding.